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The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry that is suitable to specifically map the content of autophagosomes and potentially other transient vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched ...

The crosstalk between brain infiltrating T cells and microglia in response to stroke remains elusive. Benakis et al. report that transcriptional signature of the stroke-associated microglia is reprogrammed by distinct T cell subpopulations. Engineered T cells overexpressing IL-10 administered four hours after stroke reinitiate microglial transcriptomic profile inducing a pro-regenerative environment. CD45+CD11b+ cells sorted from the ipsilateral hemisphere in naïve mice or 5 d after stroke in WT ...

Comparison of TNFα activated BMDM from B6.129P2-Apoetm1Unc/J mice treated with the inhibitors TMP195 or TPCA-1. Cells were treated for 1h with the respective inhibitors, followed by 24h stimulation with TNFα and subsequently lysed in Trizol.

pAstros were transduced with control or Ngn2-expressing retrovirus. 2DPT, media was replaced with differentiation media in absence of presence of AMG; 20DPT, DsRed-positive cells were sorted, barcoded with CMOs and analyzed by scRNAseq via 10XGenomics platform.

pAstros were transduced with control or Ngn2-expressing retrovirus. 2DPT, media was replaced with differentiation media in absence of presence of AMG; 5DPT, DsRed-positive cells were sorted, barcoded with CMOs and analyzed by scRNAseq via 10XGenomics platform

Human IPSCs from control or patients with NDUFS4 mutations were differentiated into proliferating and non-proliferating astrocytes. Three control and 3 patients lines were analyzed. Per each line, 3 biological replicates were collected at 3 stages: IPSC, proliferating astrocyte and non-proliferating astrocytes.

To determine whether CD8+ T cells contribute to oligodendrocyte and myelin pathology in 5xFAD mice, we treated 6-months old 5xFAD mice with antibodies against CD8. For the depletion of CD8+ T cells mice, 5xFAD mice aged 6 months were intraperitoneally injected with anti-CD8 antibody (BioXCell, BP0061) and their respective isotype controls (BioXCell, BE0119) twice a week for a total of 6 weeks.For 10X genomic experiments, mice were deeply anesthetized and perfused with cold PBS. Each brain was ...

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